Resistance to both RAL and EVG has been described to be associated mainly with changes at integrase positions 148 and 155. Our phenotypic results confirm the impact of these changes, since they conferred high FCs to both drugs. The Y143R mutation leads to the third RAL resistance pattern, and again, its impact on RAL susceptibility was confirmed in our study, showing high FC values. Interestingly, this mutation also impaired EVG susceptibility, showing an FC of 4 to 8 when the Y143R mutation was present along with other secondary changes. This is somewhat unexpected, since previous reports claimed that the Y143R mutation did not influence EVG susceptibility (5
). Altogether, our results suggest that EVG activity is compromised in the presence of any of the RAL resistance patterns, including the Y143R mutation. Therefore, the sequential use of these drugs is not advisable upon failure with either of them.
A few samples in our study displayed small, but potentially clinically significant, reductions in susceptibility to integrase inhibitors, based upon the established biological cutoffs of FC values of 2.0 and 1.9 for RAL and EVG, respectively. The reduced sensitivity was more prominent for EVG in the absence of primary INI resistance mutations. One specimen from a subject failing on a RAL-containing regimen did not show any known INI resistance mutation but displayed FC values of 2.5 for RAL and 7.71 for EVG. This sample harbored only the R263K mutation as being potentially responsible for this effect on INI susceptibility. Interestingly, the R263K mutation was previously shown to be selected in vitro
by EVG, conferring an FC of 5.2 to the drug (10
). In the present study, this mutation also led to a slight but significant decrease in RAL susceptibility. Another patient displayed an FC of 2.7 for RAL despite lacking primary INI resistance mutations. However, the L74I mutation had been selected in this patient failing on a RAL-containing regimen. Given that the L74M mutation is a secondary resistance mutation generally selected along with the N155H mutation in patients failing on RAL-containing regimens (8
), we suggest that the L74I mutation might equally compromise RAL susceptibility.
Overall, the testing of samples collected from patients with RAL failures in the absence of primary INI resistance mutations allowed the recognition of certain minor changes that might impact INI susceptibility in a clinically relevant manner. This was the case for the S24G/N, D25E, L74I, K215N, and Q221H/S changes for RAL and the L45Q/V, V72I, and R263K changes for EVG. Additional resistance studies are being performed by Virco to clear up the phenotypic effect of each of these changes. However, their clinical impact should be further investigated with larger groups of patients experiencing INI failures.
Most patients displaying resistance to RAL in our study also exhibited a reduced EVG susceptibility. The extent of cross-resistance was high for all different resistance patterns and may have important clinical implications regarding the consideration of the sequential use of RAL and EVG. Moreover, it was intriguing that EVG showed increased FC values more often than RAL, suggesting that integrase changes had a greater influence on EVG. Hypothetically, our findings could be explained by the faster time of dissociation from the viral integrase for EVG than for RAL, with half-lives of 2.4 and 9 h, respectively (9
The variability at the viral integrase has been shown to be greater in antiretroviral-experienced than in drug-naïve patients (1
). All our patients, regardless of RAL exposure, were antiretroviral experienced, which might have led to a higher rate of polymorphisms, providing an explanation of the relatively high proportion of subjects with slight reductions in RAL and especially EVG susceptibilities in INI-naïve patients.
In summary, our results for the testing of phenotypic and genotypic samples from patients with RAL failures and controls support that susceptibility to RAL and EVG is impaired mainly by the Q148H/R, N155H, and/or Y143R resistance mutation. Slight reductions in susceptibility to any of these drugs can occur in the presence of other integrase changes. The wide extent of cross-resistance between RAL and EVG should preclude their sequential use.